Methods of treating rheumatoid arthritis

ABSTRACT

Imidazole-based compounds, compositions comprising them, and methods of their use for the treatment, prevention and management of inflammatory and autoimmune diseases and disorders are disclosed. Particular compounds are of formula I:

This is a continuation of U.S. patent application Ser. No. 11/698,253,filed Jan. 25, 2007, which claims priority to U.S. provisionalapplication Nos. 60/815,221, filed Jun. 20, 2006, and 60/776,473, filedFeb. 24, 2006, all of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

This invention relates to imidazole-based compounds, and methods oftheir use for the treatment, prevention and management of variousdiseases and disorders.

2. BACKGROUND

Sphingosine-1-phosphate (S1P) is a bioactive molecule with potenteffects on multiple organ systems. Saba, J. D. and Hla, T. Circ. Res.94:724-734 (2004). Although some believe the compound is anintracellular secondary messenger, its mode of action is still a subjectof debate. Id. Indeed, even its metabolism is poorly understood. Hla,T., Science 309:1682-3 (2005). Researchers currently believe that S1P isformed by the phosphorylation of sphingosine, and degraded bydephosphorylation or cleavage. Its cleavage into ethanolamine phosphateand a long-chain aldehyde is reportedly catalyzed by S1P lyase. Id.;Pyne & Pyne, Biochem J. 349:385-402 (2000).

Sphingosine-1-phosphate lyase is a vitamin B₆-dependent enzyme localizedin the membrane of the endoplasmic reticulum. Van Veldhoven andMannaerts, J. Biol. Chem. 266:12502-12507 (1991); Van Veldhoven andMannaerts, Adv. Lipid. Res. 26:69 (1993). The polynucleotide and aminoacid sequences of human SP1 lyase and its gene products are described inPCT Patent Application No. WO 99/16888.

Recently, Schwab and coworkers concluded that a component of caramelcolor III, 2-acetyl-4-tetrahydroxybutylimidazole (THI), inhibits S1Plyase activity when administered to mice. Schwab, S. et al., Science309:1735-1739 (2005). While others have postulated that THI exerts itseffects by a different mechanism (see, e.g., Pyne, S. G., ACGC Chem.Res. Comm. 11:108-112 (2000)), it is clear that administration of thecompound to rats and mice induces lymphopenia and causes theaccumulation of mature T cells in the thymus. See, e.g., Schwab, supra;Pyne, S. G., ACGC Chem. Res. Comm. 11:108-112 (2000); Gugsyan, R., etal., Immunology 93(3):398-404 (1998); Halweg, K. M. and Büchi, G., J.Org. Chem. 50:1134-1136 (1985); U.S. Pat. No. 4,567,194 to Kroeplien andRosdorfer. Still, there are no known reports of THI having animmunological effect in animals other than mice and rats. Although U.S.Pat. No. 4,567,194 alleges that THI and some related compounds may beuseful as immunosuppressive medicinal agents, studies of the compound inhumans found no immunological effects. See Thuvander, A. and Oskarsson,A., Fd. Chem. Toxic. 32(1):7-13 (1994); Houben, G. F., et al., Fd. Chem.Toxic. 30(9):749-757 (1992).

3. SUMMARY OF THE INVENTION

This invention is directed, in part, to compounds of formula I:

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof, wherein: X is O or NR₃; R₁ is OR_(1A), NHOH, hydrogen, oroptionally substituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl,heterocycle, alkylheterocycle, or heterocyclealkyl; R₂ is OR_(2A),C(O)OR_(2A), hydrogen, halogen, nitrile, or optionally substitutedalkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; R₃ is OR_(3A), N(R_(3A))₂,NHC(O)R_(3A), NHSO₂R_(3A), or hydrogen; R₄ is OR_(4A), OC(O)R_(4A),hydrogen, halogen, or optionally substituted alkyl, aryl, alkylaryl,arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl; R₅ is N(R_(5A))₂, hydrogen, hydroxy, or optionallysubstituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; and each of R_(1A), R_(2A),R_(3A), R_(4A), and R_(5A) is independently hydrogen or optionallysubstituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl.

This invention also encompasses pharmaceutical compositions comprisingcompounds of formula I, and methods of treating inflammatory diseasesand disorders using compounds of formula I.

4. BRIEF DESCRIPTION OF THE FIGURES

Certain aspects of this invention can be understood with reference tothe following figures:

FIG. 1 provides a representative flow cytometry (FACS) dot plot showingthe effect of vehicle control (VC), a compound of the invention(Compound 1), and THI on T cell composition (CD4+ and CD8+) of thymus,and the mean/Sd for all mice (n=3).

FIG. 2 provides a representative FACS dot plot showing the effect ofvehicle control (VC), a compound of the invention (Compound 1), and THIon a subset of CD4+ cells (recent thymic emigrants) in mice.

FIG. 3 provides a representative FACS dot plot showing the effect ofvehicle control (VC), a compound of the invention (Compound 1), and THIon a subset of CD8+ cells (recent thymic emigrants) in mice.

FIG. 4 shows the effects of vehicle control, THI, Compound 1, andCompound 2 on whole blood counts of mice.

FIG. 5 shows the effect of single oral dosing of a compound of theinvention on collage-induced arthritis in mice.

FIG. 6 shows the effect of single oral dosing of a compound of theinvention on white blood cell and lymphocyte counts of cynomolgusmonkeys.

5. DETAILED DESCRIPTION

This invention results, in part, from studies of S1P lyasegene-disrupted mice, and largely relates to compounds believed to beuseful in the treatment, prevention and/or management of autoimmune andinflammatory diseases and disorders.

5.1. Definitions

Unless otherwise indicated, the term “alkenyl” means a straight chain,branched and/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or2 to 6) carbon atoms, and including at least one carbon-carbon doublebond. Representative alkenyl moieties include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl and3-decenyl.

Unless otherwise indicated, the term “alkyl” means a straight chain,branched and/or cyclic (“cycloalkyl”) hydrocarbon having from 1 to 20(e.g., 1 to 10 or 1 to 4) carbon atoms. Alkyl moieties having from 1 to4 carbons are referred to as “lower alkyl.” Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyland dodecyl. Cycloalkyl moieties may be monocyclic or multicyclic, andexamples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andadamantyl. Additional examples of alkyl moieties have linear, branchedand/or cyclic portions (e.g., 1-ethyl-4-methyl-cyclohexyl). The term“alkyl” includes saturated hydrocarbons as well as alkenyl and alkynylmoieties.

Unless otherwise indicated, the term “alkylaryl” or “alkyl-aryl” meansan alkyl moiety bound to an aryl moiety.

Unless otherwise indicated, the term “alkylheteroaryl” or“alkyl-heteroaryl” means an alkyl moiety bound to a heteroaryl moiety.

Unless otherwise indicated, the term “alkylheterocycle” or“alkyl-heterocycle” means an alkyl moiety bound to a heterocycle moiety.

Unless otherwise indicated, the term “alkynyl” means a straight chain,branched or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2to 6) carbon atoms, and including at least one carbon-carbon triplebond. Representative alkynyl moieties include acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl,6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl,8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyl.

Unless otherwise indicated, the term “alkoxy” means an —O-alkyl group.Examples of alkoxy groups include, but are not limited to, —OCH₃,—OCH₂CH₃, —O(CH₂)₂CH₃, —O(CH₂)₃CH₃, —O(CH₂)₄CH₃, and —O(CH₂)₅CH₃.

Unless otherwise indicated, the term “aryl” means an aromatic ring or anaromatic or partially aromatic ring system composed of carbon andhydrogen atoms. An aryl moiety may comprise multiple rings bound orfused together. Examples of aryl moieties include, but are not limitedto, anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl,naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, andtolyl.

Unless otherwise indicated, the term “arylalkyl” or “aryl-alkyl” meansan aryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term “circulating lymphocyte reductionagent” means a compound that has a CLRF of greater than about 20percent.

Unless otherwise indicated, the term “circulating lymphocyte reductionfactor” or “CLRF” means the decrease in the number of circulatinglymphocytes in mice caused by oral administration of a single dose of acompound at 100 mg/kg, as determined by the method described in theExamples, below.

Unless otherwise indicated, the terms “halogen” and “halo” encompassfluorine, chlorine, bromine, and iodine.

Unless otherwise indicated, the term “heteroalkyl” refers to an alkylmoiety (e.g., linear, branched or cyclic) in which at least one of itscarbon atoms has been replaced with a heteroatom (e.g., N, O or S).

Unless otherwise indicated, the term “heteroaryl” means an aryl moietywherein at least one of its carbon atoms has been replaced with aheteroatom (e.g., N, O or S). Examples include, but are not limited to,acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl,benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl,imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl,phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl,and triazinyl.

Unless otherwise indicated, the term “heteroarylalkyl” or“heteroaryl-alkyl” means a heteroaryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term “heterocycle” refers to anaromatic, partially aromatic or non-aromatic monocyclic or polycyclicring or ring system comprised of carbon, hydrogen and at least oneheteroatom (e.g., N, O or S). A heterocycle may comprise multiple (i.e.,two or more) rings fused or bound together. Heterocycles includeheteroaryls. Examples include, but are not limited to,benzo[1,3]dioxolyl, 2,3-dihydro-benzo[1,4]dioxinyl, cinnolinyl, furanyl,hydantoinyl, morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl,pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl and valerolactamyl.

Unless otherwise indicated, the term “heterocyclealkyl” or“heterocycle-alkyl” refers to a heterocycle moiety bound to an alkylmoiety.

Unless otherwise indicated, the term “heterocycloalkyl” refers to anon-aromatic heterocycle.

Unless otherwise indicated, the term “heterocycloalkylalkyl” or“heterocycloalkyl-alkyl” refers to a heterocycloalkyl moiety bound to analkyl moiety.

Unless otherwise indicated, the terms “manage,” “managing” and“management” encompass preventing the recurrence of the specifieddisease or disorder in a patient who has already suffered from thedisease or disorder, and/or lengthening the time that a patient who hassuffered from the disease or disorder remains in remission. The termsencompass modulating the threshold, development and/or duration of thedisease or disorder, or changing the way that a patient responds to thedisease or disorder.

Unless otherwise indicated, the term “pharmaceutically acceptable salts”refers to salts prepared from pharmaceutically acceptable non-toxicacids or bases including inorganic acids and bases and organic acids andbases. Suitable pharmaceutically acceptable base addition salts include,but are not limited to, metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Suitable non-toxic acids include, but are not limited to,inorganic and organic acids such as acetic, alginic, anthranilic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic,glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phenylacetic, phosphoric, propionic, salicylic, stearic, succinic,sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid.Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric,sulfuric, and methanesulfonic acids. Examples of specific salts thusinclude hydrochloride and mesylate salts. Others are well-known in theart. See, e.g., Remington's Pharmaceutical Sciences (18th ed., MackPublishing, Easton Pa.: 1990) and Remington: The Science and Practice ofPharmacy (19th ed., Mack Publishing, Easton Pa.: 1995).

Unless otherwise indicated, the terms “prevent,” “preventing” and“prevention” contemplate an action that occurs before a patient beginsto suffer from the specified disease or disorder, which inhibits orreduces the severity of the disease or disorder. In other words, theterms encompass prophylaxis.

Unless otherwise indicated, a “prophylactically effective amount” of acompound is an amount sufficient to prevent a disease or condition, orone or more symptoms associated with the disease or condition, orprevent its recurrence. A prophylactically effective amount of acompound means an amount of therapeutic agent, alone or in combinationwith other agents, which provides a prophylactic benefit in theprevention of the disease. The term “prophylactically effective amount”can encompass an amount that improves overall prophylaxis or enhancesthe prophylactic efficacy of another prophylactic agent.

Unless otherwise indicated, the term “S1P level enhancing agent” means acompound that has a SLEF of at least about 10-fold.

Unless otherwise indicated, the term “S1P level enhancing factor” or“SLEF” means the increase in S1P in the spleens of mice caused by oraladministration of a single dose of a compound at 100 mg/kg, asdetermined by the method described in the Examples, below. Unlessotherwise indicated, the term “stereoisomeric mixture” encompassesracemic mixtures as well as stereomerically enriched mixtures (e.g.,R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35 and 70/30).

Unless otherwise indicated, the term “stereomerically pure” means acomposition that comprises one stereoisomer of a compound and issubstantially free of other stereoisomers of that compound. For example,a stereomerically pure composition of a compound having one stereocenterwill be substantially free of the opposite stereoisomer of the compound.A stereomerically pure composition of a compound having twostereocenters will be substantially free of other diastereomers of thecompound. A typical stereomerically pure compound comprises greater thanabout 80% by weight of one stereoisomer of the compound and less thanabout 20% by weight of other stereoisomers of the compound, greater thanabout 90% by weight of one stereoisomer of the compound and less thanabout 10% by weight of the other stereoisomers of the compound, greaterthan about 95% by weight of one stereoisomer of the compound and lessthan about 5% by weight of the other stereoisomers of the compound,greater than about 97% by weight of one stereoisomer of the compound andless than about 3% by weight of the other stereoisomers of the compound,or greater than about 99% by weight of one stereoisomer of the compoundand less than about 1% by weight of the other stereoisomers of thecompound.

Unless otherwise indicated, the term “substituted,” when used todescribe a chemical structure or moiety, refers to a derivative of thatstructure or moiety wherein one or more of its hydrogen atoms issubstituted with a chemical moiety or functional group such as, but notlimited to, alcohol, aldehylde, alkoxy, alkanoyloxy, alkoxycarbonyl,alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl,alkylcarbonyloxy (—OC(O)alkyl), amide (—C(O)NH-alkyl- or-alkylNHC(O)alkyl), amidinyl (—C(NH)NH-alkyl or —C(NR)NH₂), amine(primary, secondary and tertiary such as alkylamino, arylamino,arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (—NHC(O)O-alkyl-or —OC(O)NH-alkyl), carbamyl (e.g., CONH₂, as well as CONH-alkyl,CONH-aryl, and CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid,carboxylic acid anhydride, carboxylic acid chloride, cyano, ester,epoxide, ether (e.g., methoxy, ethoxy), guanidino, halo, haloalkyl(e.g., —CCl₃, —CF₃, —C(CF₃)₃), heteroalkyl, hemiacetal, imine (primaryand secondary), isocyanate, isothiocyanate, ketone, nitrile, nitro, oxo,phosphodiester, sulfide, sulfonamido (e.g., SO₂NH₂), sulfone, sulfonyl(including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl),sulfoxide, thiol (e.g., sulfhydryl, thioether) and urea(—NHCONH-alkyl-).

Unless otherwise indicated, a “therapeutically effective amount” of acompound is an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or condition, or to delay orminimize one or more symptoms associated with the disease or condition.A therapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapies, whichprovides a therapeutic benefit in the treatment or management of thedisease or condition. The term “therapeutically effective amount” canencompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of a disease or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

Unless otherwise indicated, the terms “treat,” “treating” and“treatment” contemplate an action that occurs while a patient issuffering from the specified disease or disorder, which reduces theseverity of the disease or disorder, or retards or slows the progressionof the disease or disorder.

Unless otherwise indicated, the term “include” has the same meaning as“include, but are not limited to,” and the term “includes” has the samemeaning as “includes, but is not limited to.” Similarly, the term “suchas” has the same meaning as the term “such as, but not limited to.”

Unless otherwise indicated, one or more adjectives immediately precedinga series of nouns is to be construed as applying to each of the nouns.For example, the phrase “optionally substituted alky, aryl, orheteroaryl” has the same meaning as “optionally substituted alky,optionally substituted aryl, or optionally substituted heteroaryl.”

It should be noted that a chemical moiety that forms part of a largercompound may be described herein using a name commonly accorded it whenit exists as a single molecule or a name commonly accorded its radical.For example, the terms “pyridine” and “pyridyl” are accorded the samemeaning when used to describe a moiety attached to other chemicalmoieties. Thus, the two phrases “XOH, wherein X is pyridyl” and “XOH,wherein X is pyridine” are accorded the same meaning, and encompass thecompounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.

It should also be noted that if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold ordashed lines, the structure or the portion of the structure is to beinterpreted as encompassing all stereoisomers of it. Moreover, any atomshown in a drawing with unsatisfied valences is assumed to be attachedto enough hydrogen atoms to satisfy the valences. In addition, chemicalbonds depicted with one solid line parallel to one dashed line encompassboth single and double (e.g., aromatic) bonds, if valences permit.

5.2. Compounds

This invention encompasses compounds of formula I:

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof, wherein: X is O or NR₃; R₁ is OR_(1A), NHOH, hydrogen, oroptionally substituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl,heterocycle, alkylheterocycle, or heterocyclealkyl; R₂ is OR_(2A),C(O)OR_(2A), hydrogen, halogen, nitrile, or optionally substitutedalkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; R₃ is OR_(3A), N(R_(3A))₂,NHC(O)R_(3A), NHSO₂R_(3A), or hydrogen; R₄ is OR_(4A), OC(O)R_(4A),hydrogen, halogen, or optionally substituted alkyl, aryl, alkylaryl,arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl; R₅ is N(R_(5A))₂, hydrogen, hydroxy, or optionallysubstituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; and each of R_(1A), R_(2A),R_(3A), R_(4A), and R_(5A) is independently hydrogen or optionallysubstituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl.

Particular compounds of formula I are such that if X is O; R₁ is alkylof 1 to 4 carbons, phenyl, benzyl or phenylethyl; R₂ is hydrogen; andone of R₄ and R₅ is hydroxyl; the other of R₄ and R₅ is not alkyl of 1to 6 carbons, hydroxyalkyl of 1 to 6 carbons, polyhydroxyalkyl of 1 to 6carbons having up to one hydroxyl per carbon, polyacetylalkyl of 1 to 6carbons having up to one acetyl per carbon, phenyl, benzyl orphenylethyl.

In particular embodiments, the compound is not2-acetyl-4-tetrahydroxybutylimidazole,1-(4-(1,1,2,2,4-pentahydroxybutyl)-1H-imidazol-2-yl)ethanone,1-(2-acetyl-1H-imidazol-4-yl)butane-1,1,2,2-tetrayl tetraacetate, or1-(2-acetyl-1H-imidazol-4-yl)butane-1,1,2,2,4-pentayl pentaacetate.

A particular embodiment encompasses compounds of formula II:

and pharmaceutically acceptable salts and solvates thereof, wherein: Xis O or NR₃; R₁ is OR_(1A), NHOH, hydrogen, or optionally substitutedalkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; R₂ is OR_(2A), C(O)OR_(2A),hydrogen, halogen, nitrile, or optionally substituted alkyl, aryl,alkylaryl, arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl; R₃ is OR_(3A), N(R_(3A))₂, NHC(O)R_(3A), NHSO₂R_(3A),or hydrogen; R₆ is OR_(6A), OC(O)R_(6A), N(R_(6B))₂, NHC(O)R_(6B),hydrogen, or halogen; R₇ is OR_(7A), OC(O)R_(7A), N(R_(7B))₂,NHC(O)R_(7B), hydrogen, or halogen; R₈ is OR_(8A), OC(O)R_(8A),N(R_(8B))₂, NHC(O)R_(8B), hydrogen, or halogen; R₉ is CH₂OR_(9A),CH₂OC(O)R_(9A), N(R_(9B))₂, NHC(O)R_(9B), hydrogen, or halogen; each ofR_(1A), R_(2A), R_(3A), R_(6A), R_(7A), R_(8A) and R_(9A) isindependently hydrogen or optionally substituted alkyl, aryl, alkylaryl,arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl; and each of R_(6B), R_(7B), R_(8B) and R_(9B) isindependently hydrogen or alkyl optionally substituted with one or morehydroxy or halogen groups;

Particular compounds of formula II are such that: 1) if X is O, R₁ isalkyl of 1 to 4 carbons, phenyl, benzyl or phenylethyl, and R₂ ishydrogen, at least two of R₆, R₇, R₈ and R₉ are not hydroxyl or acetate;2) if X is O, R₁ is methyl, R₂ is hydrogen, R₆ and R₇ are both hydroxyl,and one of R₈ and R₉ is hydrogen, the other is not NHC(O)R_(9B); 3) if Xis O, R₁ is OR_(1A), R_(1A) is hydrogen or lower alkyl, and R₂ ishydrogen, at least one, but not all, of R₆, R₇, R₈ and R₉ is hydroxyl oracetate.

Particular compounds of the invention are of formula II(a):

Others are of formula III:

wherein: Z is optionally substituted alkyl; R₁ is OR_(1A), NHOH,hydrogen, or optionally substituted alkyl, aryl, alkylaryl, arylalkyl,heteroalkyl, heterocycle, alkylheterocycle, or heterocyclealkyl; R₂ isOR_(2A), C(O)OR_(2A), hydrogen, halogen, nitrile, or optionallysubstituted alkyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heterocycle,alkylheterocycle, or heterocyclealkyl; R₃ is OR_(3A), N(R_(3A))₂,NHC(O)R_(3A), NHSO₂R_(3A), or hydrogen; and each of R_(1A), R_(2A), andR_(3A) is independently hydrogen or optionally substituted alkyl, aryl,alkylaryl, arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl.

Another embodiment of the invention encompasses compounds of formula IV:

and pharmaceutically acceptable salts and solvates thereof, wherein: R₁is OR_(1A), NHOH, hydrogen, or optionally substituted alkyl, aryl,alkylaryl, arylalkyl, heteroalkyl, heterocycle, alkylheterocycle, orheterocyclealkyl; R₃ is OR_(3A), N(R_(3A))₂, NHC(O)R_(3A), NHSO₂R_(3A),or hydrogen; R₆ is OR_(6A), OC(O)R_(6A), N(R_(6B))₂, NHC(O)R_(6B),hydrogen, or halogen; R₇ is OR_(7A), OC(O)R_(7A), N(R_(7B))₂,NHC(O)R_(7B), hydrogen, or halogen; R₈ is OR_(8A), OC(O)R_(8A),N(R_(8B))₂, NHC(O)R_(8B), hydrogen, or halogen; R₉ is CH₂OR_(9A),CH₂OC(O)R_(9A), N(R_(9B))₂, NHC(O)R_(9B), hydrogen, or halogen; and eachof R_(1A), R_(3A), R_(6A), R_(7A), R_(8A) and R_(9A) is independentlyhydrogen or optionally substituted alkyl, aryl, alkylaryl, arylalkyl,heteroalkyl, heterocycle, alkylheterocycle, or heterocyclealkyl.

Particular compounds are of formula IV(a):

With regard to each of the formulae shown above that contain themoieties described below, certain embodiments of the invention are suchthat:

In some, X is O. In others, X is NR₃.

In some, R₁ is hydrogen. In others, R₁ is optionally substituted loweralkyl. In others, R₁ is NHOH. In others, R₁ is OR_(1A) and R_(1A) is,for example, hydrogen or optionally substituted lower alkyl.

In some, R₂ is hydrogen. In others, R₂ is not hydrogen. In others, R₂ isnitrile. In others, R₂ is optionally substituted lower alkyl. In others,R₂ is OR_(2A). In others, R₂ is C(O)OR_(2A). In some, R_(2A) is hydrogenor optionally substituted lower alkyl.

In some, R₃ is OR_(3A). In others, R₃ is N(R_(3A))₂ or NHC(O)R_(3A). Inothers, R₃ is NHSO₂R_(3A). In some, R_(3A) is hydrogen or optionallysubstituted lower alkyl. In others, R_(3A) is optionally substitutedaryl or heterocycle.

In some, R₄ is OR_(4A). In others, R₄ is halogen.

In some, R₅ is N(R_(5A))₂. In others, R₅ is hydrogen. In others, R₅ ishydroxyl. In others, R₅ is heteroalkyl (e.g., alkoxy). In others, R₅ isoptionally substituted alkyl. In others, R₅ is optionally substitutedaryl.

In some, one or more of R₆, R₇, R₈, and R₉ is hydroxy or halogen. Insome, all of R₆, R₇, R₈, and R₉ are hydroxyl or acetate.

In some, Z is alkyl optionally substituted with one or more hydroxyl,acetate or halogen moieties.

Compounds of the invention may contain one or more stereocenters, andcan exist as racemic mixtures of enantiomers or mixtures ofdiastereomers. This invention encompasses stereomerically pure forms ofsuch compounds, as well as mixtures of those forms. Stereoisomers may beasymmetrically synthesized or resolved using standard techniques such aschiral columns or chiral resolving agents. See, e.g., Jacques, J., etal., Enantiomers, Racemates and Resolutions (Wiley Interscience, NewYork, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); andWilen, S. H., Tables of Resolving Agents and Optical Resolutions, p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

This invention further encompasses stereoisomeric mixtures of compoundsdisclosed herein. It also encompasses configurational isomers ofcompounds disclosed herein, either in admixture or in pure orsubstantially pure form, such as cis (Z) and trans (E) alkene isomersand syn and anti oxime isomers.

Preferred compounds of the invention are circulating lymphocytereduction agents. Certain compounds inhibit the amount of circulatinglymphocytes, as determined using the method described in the Examples,by greater than about 20, 50, 75, 100, 150 or 200 percent. In thisregard, it has been found that while THI is capable of reducingcirculating lymphocytes in mice, many analogues and derivatives of THI,such as1-(4-methyl-5-((1S,2R,3R)-1,2,3,4-tetrahydroxybutyl)thiazol-2-yl)ethanone,have little or no effect on circulating lymphocytes, despite reports tothe contrary. See WO 97/46543.

Without being limited by theory, compounds of the invention are believedto affect the S1P metabolic pathway, and may inhibit S1P lyase directlyor indirectly in vivo. Particular compounds are S1P level enhancingagents. Certain compounds increase the amount of S1P, as determinedusing the method described below in the Examples, by greater than about10, 15, 20, 25, or 30-fold.

Compounds of the invention can be prepared by methods known in the art(e.g., by varying and adding to the approaches described in Pyne, S. G.,ACGC Chem. Res. Comm. 11:108-112 (2000); Halweg, K. M. and Büchi, G., J.Org. Chem. 50:1134-1136 (1985)). Compounds can also be made by themethods disclosed below and variants thereof, which will be apparent tothose of ordinary skill in the art.

5.3. Methods of Use

This invention encompasses a method of modulating (e.g., increasing) theamount of S1P in a patient (e.g., a mouse, rat, dog, cat or human) inneed thereof, which comprises administering to the patient an effectiveamount of a compound of the invention (i.e., a compound disclosedherein).

Another embodiment encompasses a method of reducing the number ofT-cells in the blood of a patient, which comprises administering to thepatient an effective amount of a compound of the invention.

Another embodiment encompasses a method of treating, managing orpreventing a disease affected by (or having symptoms affected by) S1Plevels, which comprises administering to a patient in need thereof atherapeutically or prophylactically effective amount of a compound ofthe invention.

Another embodiment encompasses a method of suppressing immune responsein a patient, which comprises administering to the patient an effectiveamount of a compound of the invention.

Another embodiment encompasses a method of treating, managing orpreventing an autoimmune or inflammatory disease or disorder, whichcomprises administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of a compound of the invention.Examples of diseases and disorders include ankylosing spondylitis,asthma (e.g., bronchial asthma), atopic dermatitis, Behcet's disease,graft-vs-host disease, Kawasaki syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, pollinosis, psoriasis, psoriaticarthritis, rheumatoid arthritis, scleroderma, transplant rejection(e.g., of organ, cell or bone marrow), type 1 diabetes, and uveitis.

Additional diseases and disorders include Addison's Disease,anti-phospholipid syndrome, autoimmune atrophic gastritis, achlorhydraautoimmune, Celiac Disease, Crohn's Disease, Cushing's Syndrome,dermatomyositis, Goodpasture's Syndrome, Grave's Disease, Hashimoto'sthyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia,Lambert-Eaton Syndrome, pemphigoid, pemphigus vulgaris, perniciousanemia, polyarteritis nodosa, primary biliary cirrhosis, primarysclerosing cholangitis, Raynauds, Reiter's Syndrome, relapsingpolychondritis, Schmidt's Syndrome, Sjogren's Syndrome, sympatheticophthalmia, Takayasu's Arteritis, temporal arteritis, thyrotoxicosis,ulcerative colitis, and Wegener's granulomatosis.

The amount, route of administration and dosing schedule of a compoundwill depend upon factors such as the specific indication to be treated,prevented, or managed, and the age, sex and condition of the patient.The roles played by such factors are well known in the art, and may beaccommodated by routine experimentation. In a particular embodiment, acompound of the invention is administered to a human patient in anamount of about 0.5, 1, 2.5 or 5 mpk.

5.4. Pharmaceutical Formulations

This invention encompasses pharmaceutical compositions comprising one ormore compounds of the invention. Certain pharmaceutical compositions aresingle unit dosage forms suitable for oral, mucosal (e.g., nasal,sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, bolus injection, intramuscular, or intraarterial), ortransdermal administration to a patient. Examples of dosage formsinclude, but are not limited to: tablets; caplets; capsules, such assoft elastic gelatin capsules; cachets; troches; lozenges; dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or awater-in-oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

The formulation should suit the mode of administration. For example,oral administration requires enteric coatings to protect the compoundsof this invention from degradation within the gastrointestinal tract.Similarly, a formulation may contain ingredients that facilitatedelivery of the active ingredient(s) to the site of action. For example,compounds may be administered in liposomal formulations, in order toprotect them from degradative enzymes, facilitate transport incirculatory system, and effect delivery across cell membranes tointracellular sites.

Similarly, poorly soluble compounds may be incorporated into liquiddosage forms (and dosage forms suitable for reconstitution) with the aidof solubilizing agents, emulsifiers and surfactants such as, but notlimited to, cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin,Captisol®, and Encapsin™ (see, e.g., Davis and Brewster, 2004, Nat. Rev.Drug Disc. 3:1023-1034), Labrasol®, Labrafil®, Labrafac®, cremafor, andnon-aqueous solvents, such as, but not limited to, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acidesters of sorbitan, and mixtures thereof (e.g., DMSO:cornoil).

Poorly soluble compounds may also be incorporated into suspensions usingother techniques known in the art. For example, nanoparticles of acompound may be suspended in a liquid to provide a nanosuspension (see,e.g., Rabinow, 2004, Nature Rev. Drug Disc. 3:785-796). Nanoparticleforms of compounds described herein may be prepared by the methodsdescribed in U.S. Patent Publication Nos. 2004-0164194, 2004-0195413,2004-0251332, 2005-0042177 A1, 2005-0031691 A1, and U.S. Pat. Nos.5,145,684, 5,510,118, 5,518,187, 5,534,270, 5,543,133, 5,662,883,5,665,331, 5,718,388, 5,718,919, 5,834,025, 5,862,999, 6,431,478,6,742,734, 6,745,962, the entireties of each of which are incorporatedherein by reference. In one embodiment, the nanoparticle form comprisesparticles having an average particle size of less than about 2000 nm,less than about 1000 nm, or less than about 500 nm.

The composition, shape, and type of a dosage form will vary depending onits use. For example, a dosage form used in the acute treatment of adisease may contain larger amounts of one or more of the activeingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Theseand other ways in which specific dosage forms encompassed by thisinvention will vary from one another will be readily apparent to thoseskilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18thed., Mack Publishing, Easton Pa. (1990).

5.4.1. Oral Dosage Forms

Pharmaceutical compositions of the invention suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, EastonPa. (1990).

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms. If desired, tablets can becoated by standard aqueous or nonaqueous techniques. Such dosage formscan be prepared by conventional methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary. Disintegrants may beincorporated in solid dosage forms to facility rapid dissolution.Lubricants may also be incorporated to facilitate the manufacture ofdosage forms (e.g., tablets).

5.4.2. Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are specifically sterileor capable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5.4.3. Transdermal, Topical and Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990);and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985). Transdermal dosage forms include “reservoir type”or “matrix type” patches, which can be applied to the skin and worn fora specific period of time to permit the penetration of a desired amountof active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms are well known to those skilled in the pharmaceutical arts, anddepend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers may be used to assist in delivering active ingredients to thetissue.

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates may also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

6. EXAMPLES

Aspects of this invention can be understood from the following examples,which do not limit its scope.

6.1. S1P Lysase Gene-Disrupted Mice

Gene trapping is a method of random insertional mutagenesis that uses afragment of DNA coding for a reporter or selectable marker gene as amutagen. Gene trap vectors have been designed to integrate into intronsor exons in a manner that allows the cellular splicing machinery tosplice vector encoded exons to cellular mRNAs. Gene trap vectorstypically contain selectable marker sequences that are preceded bystrong splice acceptor sequences and are not preceded by a promoter.Thus, when such vectors integrate into a gene, the cellular splicingmachinery splices exons from the trapped gene onto the 5′ end of theselectable marker sequence. Typically, such selectable marker genes canonly be expressed if the vector encoding the gene has integrated into anintron. The resulting gene trap events are subsequently identified byselecting for cells that can survive selective culture.

Embryonic stem cells (derived murine strain A129) were mutated by aprocess involving the insertion of at least a portion of a geneticallyengineered vector sequence into the SP1 lyase gene. The mutatedembryonic stem cells were then microinjected into blastocysts, whichwere subsequently introduced into pseudopregnant female hosts andcarried to term using established methods. In this case, the virusinserted between exons 1 and 2, and disrupted the SP1 lyase gene. Theresulting chimeric animals were subsequently bred to produce offspringcapable of germline transmission of an allele containing the engineeredmutation in the SP1 lyase gene.

Techniques useful to disrupt a gene in a cell, and especially an EScell, that may already have a disrupted gene are disclosed in U.S. Pat.Nos. 6,136,566; 6,139,833 and 6,207,371 and U.S. patent application Ser.No. 08/728,963, each of which are incorporated herein by reference inits entirety.

6.2. Hematological Effects of S1P Lyase Gene Disruption

Whole blood was collected by retrorbital bleed and placed in a capillaryblood collection tube that contained EDTA. The blood was analyzed usingthe Cell-Dyn 3500R analyzer (Abbott Diagnostics). The analyzer employsdual technologies to provide the basis for a five-part white blood cell(WBC) differential identification. Multi-Angle Polarized ScatterSeparation (M.A.P.S.S.) provides the primary white blood cell count anddifferential information, while impedance provides additionalinformation in the presence of fragile lymphocytes and hypotonicallyresistant red blood cells.

Approximately 135 microliters of whole blood was aspirated into theanalyzer using a peristaltic pump. Four independent measurementtechniques were used by the Cell-Dyn 3500R System (Abbott, Ill.) toobtain the hematologic parameters. The WBC Optical Count (WOC) and theWBC differential data were measured in the optical flow channel,resulting in the identification of the WBC subpopulations (neutrophils,lymphocytes, monocytes, eosinophils, and basophils) for the five partWBC differential. The WBC Impedance Count (WIC) was measured in oneelectrical impedance channel. The RBC and platelet data were measured ina second electrical impedance channel. The hemoglobin was measured inthe spectrophotometric channel. The sample was aspirated, diluted,mixed, and the measurements for each parameter were obtained during eachinstrument cycle. The final hematological analysis parameters obtainedwere white blood cell count, neutrophils, lymphocytes, monocytes,eosinophils, basophils, red blood cells, hemoglobin, hematocrit,platlets, red cell distribution width, mean corpuscular volume and meanplatelet volume.

Blood samples were obtained from a total of 16 mice. Analysis andcomparison of the blood samples were obtained from seven wild-type mice,six heterozygous mice and three homozygous mice. There was nosignificant genotype associated differences between mice from differentgroups with regard to mean red blood cell (RBC) counts, hemoglobinlevels, mean corpuscular volume, mean corpuscular hemoglobin, meancorpuscular hemoglobin concentration, platelet counts or mean plateletvolume. There were differences in hematocrit and red cell distributionwidth. The mean hematocrit in homozygous (−/−) mice was 37±2.56 percent,in heterozygous (+/−) mice, 40.9±4 percent and in wild-type (+/+) miceit was 44.7±2.7 percent. The red blood cell distribution width inhomozygous (−/−) mice was 25.2±4.2 percent, in heterozygous (+/−) mice,17.6±1.9 percent and in wild-type (+/+) mice it was 17.2±2 percent.

Similarly, SP1 lyase deficient mice had no significant differences inthe total white blood cell counts as compared to heterozygous orwild-type mice. Homozygous (−/−) mice had a total white blood cell countof 7200±700 cells/μl. Heterozygous (+/−) mice had total white blood cellcount of 6200±1800 cells/μl and wild-type (+/+) mice had a total whiteblood cell count of 7200±2600 cells/μl.

In the mice that were homozygous for disruption of SP1 lyase, lymphocytecounts was decreased, while the number of neutrophils, monocytes,eosinophils, and basophils were increased. The blood lymphocyte countsof mice homozygous(−/−) for disruption in the SP1 lyase gene was greatlyreduced. The mean lymphocyte count in homozygous (−/−) mice was 847±139cells/μl, in heterozygous (+/−) mice the mean lymphocyte count was4582±2364 cells/μl, as opposed to the mean lymphocyte count in wild-type(+/+) mice which was 6126±2151 cells/μl.

In contrast, mean neutrophil count in homozygous (−/−) mice was 5020±612cells/μl, while in heterozygous (+/−) mice the mean neutrophil count was1380±1140 cells/μl and wild-type (+/+) mice had a mean neutrophil countof only 886±479 cells/μl. Similarly, the mean monocyte count inhomozygous (−/−) mice was 950±218 cells/μl, while in heterozygous (+/−)mice the mean monocyte count was 250±108 cells/μl and in wild-type (+/+)mice the mean monocyte count was only 146±92 cells/μl. Likewise witheosinophils, the mean eosinophil count in homozygous (−/−) mice was247±297 cells/μl, while in heterozygous (+/−) mice the mean eosinophilcount was 8±8 cells/μl and in wild-type (+/+) mice the mean eosinophilcount was only 14±21 cells/μl.

The same was true of basophils. The mean basophil count in homozygous(−/−) mice was 130±90 cells/μl, in heterozygous (+/−) mice the meanbasophil count was 7±5 cells/μl and in wild-type (+/+) mice the meanbasophil count was only 16±11 cells/μl.

Similarly, the mean monocyte count in homozygous (−/−) mice was 950±218cells/μl, while in heterozygous (+/−) mice the mean monocyte count was250±108 cells/μl and in wild-type (+/+) mice the mean monocyte count wasonly 146±92 cells/μl. Likewise with eosinophils, the mean eosinophilcount in homozygous (−/−) mice was 247±297 cells/μl, while inheterozygous (+/−) mice the mean eosinophil count was 8±8 cells/μl andin wild-type (+/+) mice the mean eosinophil count was only 14±21cells/μl.

6.3. Synthesis of(E/Z)-1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)-ethanoneoxime

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(THI, prepared according to Halweg, K. M. and Büchi, G., J. Org. Chem.50:1134-1136 (1985)) (350 mg, 1.52 mmol) was suspended in water (10 ml).Hydroxylamine hydrochloride (126.8 mg, 1.82 mmol, 1.2 eq.) and sodiumacetate (247.3 mg, 3.04 mmol. 2 eq.) was added, and the suspension wasstirred at 50° C. The reaction mixture turned clear after approximately4 hours. Stirring was continued at 50° C. for 16 hours. LCMS analysisindicated the formation of the product and the absence of startingmaterial. The reaction mixture was allowed to attain room temperatureand passed through a fine porosity filter. This solution was useddirectly to purify the product by using preparative HPLC: Atlantis HILICsilica column 30×100 mm; 2%-21% water in acetonitrile over 6 minutes; 45ml/min; with detection at 254 nm. The product fractions were collectedand the acetonitrile was evaporated under reduced pressure. The aqueoussolution was lyophilized to yield the product, a mixture ofapproximately 3:1 anti:syn isomers, as a white solid: 284 mg (77%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 0-17% MeOH (0.1% TFA) in water(0.1% TFA) over 5 min; flow rate=3 ml/min; Detection 220 nm; Retentiontimes: 0.56 min (syn isomer, 246.0 (M+1)) and 0.69 min (anti isomer,246.0 (M+1)). ¹H NMR (D₂O and DCl) δ 2.15 and 2.22 (singlets, 3H),3.5±3.72 (m, 4H), 4.76 (br, OH protons and H₂O), 4.95 and 4.97(singlets, 1H), 7.17 and 7.25 (singlets, 1H). ¹³C NMR (D₂O and DCl) δ10.80, 16.76, 63.06, 64.59, 64.75, 70.86, 72.75, 72.85, 117.22, 117.64,135.32, 138.39, 141.35, 144.12.

6.4. Synthesis of(E)-1-(4-((1R,2S3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)-ethanoneoxime

This compound was prepared in two steps, as shown below

First, to a flask charged with THI (21.20 mmol, 4.88 g) is added water(25 ml) and 1N aqueous HCl (21.2 ml, 21.2 mmol). After all solidsdissolved, a solution of trityl hydroxylamine (25.44 mmol, 7.00 g) indioxane (55 ml) was added and the reaction was maintained at 50° C. for4 h. At completion, the reaction was cooled room temperature and thesolution was adjusted to pH=7 by addition of 1N aqueous NaOH. Theneutralized solution was then concentrated to a plastic mass, which waspurified by flash chromatography on silica gel [10% MeOH/1% NH₄OH (5%wt. solution in water) in DCM] to provide the trityl-ether as clearplastic. Treatment of the plastic mass with hexane and concentrationprovided a white foam, which could be dried under vacuum to a flakeysolid (10.00 g, 97% yield).

Second, to a vigorously stirred, room temperature solution of the trityloxime-ether (4.8 g, 10 mmol) in dioxane (90 ml) is added a solution ofHCl in dioxane (4M, 60 ml). After a few minutes, a white precipitant wasobserved, and stirring was continued for a total of 30 minutes, beforefiltering over a fritted glass filter and rinsing the cake with dioxaneand ether. The cake was redissolved in water (200 ml), sonicated for 5min, then cooled to 0° C., treated with celite (5 g), and filtered overa fritted glass filter. The aqueous solution was concentrated todryness, then isolated from methanol (30 ml)/diethyl ether (60 ml) toprovide the E-oxime as an analytically pure white powder (3.8 g, 80%yield).

6.5. Synthesis of(E/Z)-1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)methanoneO-methyl oxime

The captioned compound was prepared as described above in Example 6.3,by using methoxylamine hydrochloride in place of hydroxylaminehydrochloride, in 74% yield. The product was a white fluffy solid.

LCMS: Sunfire C-18 column, 4.6×50 mm; 0-17% MeOH (0.1% TFA) in water(0.1% TFA) over 5 min; flow rate=3 ml/min; Detection 220 nm; Retentiontimes: 1.59 minutes (syn isomer, 260.1 (M+1)) and 1.73 min (anti isomer,260.1 (M+1)). ¹H NMR (D₂O) δ 2.18 and 2.22 (singlets, 3H), 3.54-3.60 (m,1H), 3.66-3.79 (m, 3H), 3.94 and 3.95 (singlets, 3H), 4.76 (br, OHprotons and H₂O), 4.93 and 4.97 (singlets, 1H), 7.17 and 7.25 (singlets,1H).

¹³C NMR (D₂O) δ 11.55, 17.56, 62.32, 62.38, 62.99, 63.07, 67.09, 71.54,73.86, 119.09, 138.64, 139.79, 142.95, 144.98, 148.97.

6.6. Synthesis of1-(5-methyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)pethanone

The captioned compound was prepared in seven steps, using the processoutlined below.

4-Methylimidazole-1-dimethylaminosulfonamide (2): To a room temperaturesolution of 4-methyl imidazole 1 (3.00 g, 36.54 mmol) in toluene (200ml) was consecutively added triethylamine 5.6 ml, 40.20 mmol) andN,N-dimethylaminosulfamoyl chloride (3.9 ml, 36.54 mmol). The vessel wasstored in a 5° C. refrigerator for 48 hours, then the solids werefiltered off from the reaction and the liquor was concentrated to obtaina 2.5:1 mixture of regioisomers 2 and 2a. The crude product was purifiedby flash chromatography over silica gel (80-100% ethyl acetate:hexaneeluent) to obtain a 2:2a in a 5.5:1 mixture of regioisomers (4.31 g, 62%yield): M+1=190.1

4-Methyl-2-acetylimidazole-1-dimethylaminosulfonamide (3): To a −78° C.solution of the imidazole 2 (1.99 g, 10.54 mmol) in tetrahydrofuran (70ml) was added slowly a solution of n-BuLi in hexane (2.5M, 11.60 ml).After 40 minutes, N-methoxy-N-methylacetamide (1.30 g, 12.65 mmol) wasadded dropwise to the cooled solution. The reaction was allowed to warmto room temperature and maintained for 2 hours. At completion, thereaction was quenched by addition of saturated aqueous NH₄Cl (20 ml),then diluted with water (20 ml). The layers were separated, and theorganic layer was washed with ethyl acetate (2×30 ml). The combinedorganics were washed with brine (20 ml), then dried over MgSO₄ andconcentrated. The crude product was purified by flash chromatographyover silica (60-80% ethyl acetate:hexane eluent) to provide 3 as an oil(1.85 g, 76% yield): M+1=232.1.

4-Methyl-2-(1-(triisopropylsilyloxy)vinyl)-1-dimethylaminosulfonamide(4): To a solution of imidazole 3 (1.65 g, 7.14 mmol) in dichloromethane(45 ml) was consecutively added triethylamine (1.00 ml, 14.28 mmol) andtriisopropylsilyl trifluoromethanesulfonate (2.12 ml, 7.86 mmol). Thereaction was maintained at room temperature for 20 hours, then quenchedby the addition of saturated aqueous NaHCO₃ (20 ml). The mixture wasdiluted with water (20 ml) and the layers were separated. The aqueouslayer was washed with dichloromethane (2×20 ml) and the combinedorganics were washed with brine solution (20 ml), then dried over MgSO₄and concentrated. The resulting oil was purified by flash chromatographyover silica gel (1-2% methanol:dichloromethane eluent) to provide silylenol ether 4 as an orange oil (2.26 g, 83% yield): M+1=388.2.

Lactol (5): To a −78° C. solution of imidazole 4 (2.26 g, 5.84 mmol) intetrahydrofuran (40 ml) was slowly added a hexane solution of n-BuLi(2.5M, 3.27 ml). After 30 minutes, a solution of(−)-2,3-O-isopropylidine-D-erythronolactone (1.66 g, 10.51 mmol) intetrahydrofuran (10 ml) was added slowly to the −78° C. solution. Thereaction was maintained at −78° C. for 2 hours, then allowed to warm to0° C. before quenching the reaction by addition of saturated aqueousNH₄Cl (20 ml). The mixture was diluted with water (10 ml) and the layerswere separated. The organics were washed with ethyl acetate (2×20 ml)and the combined organics were washed with brine (20 ml), then driedover MgSO₄ and concentrated. The crude product was purified on silicagel (30-50% ethyl acetate:hexane eluent) to provide the lactol 5 (2.69g, 85% yield) as a white foam: M+1=546.4.

Diol (6): To a 0° C. solution of the lactol 5 (2.09 g, 3.83 mmol) inethanol (70 ml) was added granular NaBH₄ (1.4 g, 38.32 mmol) in a fewportions. After 2 hours, the reaction was warmed to room temperature for30 minutes, then concentrated. The residue was redissolved in water (40ml) and ethyl acetate (40 ml). The biphasic mixture was stirredvigorously for 10 minutes, then the layers were separated. The aqueouslayer was washed with ethyl acetate (2×40 ml) and the combined organicswere washed with brine (30 ml), then dried over MgSO₄ and concentrated.The crude foam was purified by flash chromatography over silica (5%methanol:dichloromethane eluent) to provide diol 6 (1.88 g, 90% yield)as a 3:1 mixture of inseparable diasteromers at the benzylic position:M+1=547.4.

Imidazole (7): Cesium fluoride (315 mg, 2.08 mmol) was added to asolution of the imidazole 6 (567 mg, 1.04 mmol) in ethanol (10 ml) andwarmed to 65° C. After 1 hour, the reaction was cooled to roomtemperature and treated with saturated aqueous NH₄Cl (1 ml), thenconcentrated. The crude product was purified by flash chromatographyover silica gel (5% methanol:dichloromethane eluent) to provideimidazole 7 (380 mg, 94% yield) as a white foam: M+1=392.1.

Final Product (8): The protected imidazole 7 (380 mg, 0.97 mmol) wasdissolved in acetone (6 ml) and consecutively treated with water (6 ml)and concentrated aqueous HCl (3 ml). The vessel was warmed to 40° C. for45 minutes, then cooled to room temperature and concentrated. The crudematerial was purified by reverse phase preparative chromatography usinga 150 mm×30 mm Zorbax C-6 column using unbuffered solvents by thefollowing method: 1% acetonitrile:water isocratic run for 5 minutes(T_(R)=1.52 minutes). Following lyophylization, compound 8 was obtainedas the dimethylaminosulfamic acid salt an amorphous solid: M+1=245.1; ¹HNMR (400 MHz, CDCl₃) major δ 5.04 (d, 1H), 3.62 (comp. m, 2H), 3.42(comp. m, 2H), 2.62 (s, 6H), 2.43 (s, 3H), 2.21 (s, 3H); minor δ 5.01(d, 1H), 3.79 (comp. m, 2H), 3.55 (comp. m, 2H), 2.62 (s, 6H), 2.43 (s,3H), 2.21 (s, 3H).

6.7. Synthesis of(1R,2S,3R)-1-(2-(1-hydrazonoethyl)-1H-imidazol-4-yl)butane-1,2,3,4-tetraol

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(THI, prepared according to Halweg, K. M. and Büchi, G., J. Org. Chem.50:1134-1136 (1985)) (148 mg, 0.64 mmol) was suspended in methanol (3ml) and water (1 ml). Hydrazine hydrate (35 mg, 0.7 mmol, 1.2 eq.) andacetic acid (one drop) were added, and the suspension was stirred at 50°C. for 6 hours. LCMS analysis indicated the formation of the product andthe absence of starting material. The reaction mixture was cooled toroom temperature and diluted with tetrahydrofuran. The resulting whiteprecipitate was collected and washed with tetrahydrofuran to yield theproduct, a mixture of approximately 3:1 E:Z isomers, as a white solid:90 mg (58%).

LCMS: Zorbax C-8 column, 4.6×150 mm; 10-90% in water (10 mM ammoniumacetate) over 6 min; flow rate=2 ml/min; Detection 220 nm; Retentiontimes: 0.576 min (syn isomer, 245.0 (M+1)) and 1.08 min (anti isomer,245.0 (M+1)). ¹H NMR (DMSO-d6) δ 2.5 (singlet, 3H under DMSO), 3.4-3.7(m, 4H), 4.3 (m, 2H), 4.6 (m, 2H), 4.8 (m, 1H), 4.9 and 5.0 (doublets,1H), 7.04 and 7.21 (singlets, 1H).

6.8. Synthesis ofN′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)acetohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(160 mg, 0.70 mmol) was suspended in methanol (3 ml) and water (1 ml).Acetic hydrazide (56 mg, 0.75 mmol, 1.2 eq.) and hydrochloric acid (onedrop, 12 N) were added, and the suspension was stirred at 50° C. for 48hours. LCMS analysis indicated the formation of the product and theabsence of starting material. The reaction mixture was cooled to roomtemperature and diluted with tetrahydrofuran. The resulting whiteprecipitate was collected and washed with tetrahydrofuran to yield theproduct, a mixture of approximately 3:1 E:Z isomers, as a white solid:129 mg (65%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 2-20% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.53 min (287.1 (M+1)). ¹H NMR (DMSO-d6) δ 2.2 (singlets, 3H), 2.5(singlets, 3H under DMSO), 3.4-3.7 (m, 4H), 4.3 (br, 2H), 4.6-5.0 (br,4H), 7.0 (br, 1H), 10.30 and 10.37 (singlets, 1H).

6.9. Synthesis of(E)-4-methyl-N′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)benenesulfonohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(153 mg, 0.67 mmol) was suspended in methanol (3 ml) and water (1 ml).P-toluenesulfonyl hydrazide (140 mg, 0.75 mmol, 1.2 eq.) andhydrochloric acid (one drop, 12 N) were added, and the suspension wasstirred at 50° C. for 24 hours. LCMS analysis indicated the formation ofthe product and the absence of starting material. The reaction mixturewas cooled to room temperature and dry-loaded on silica gel. Flashchromatography on silica gel (10 g SiO₂, 4:1 ethyl acetate:methanol) toyield the product, a mixture of approximately 85:15 E:Z isomers, as awhite solid: 142 mg (53%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontimes: 0.50 min (399.2 (M+1)) and 0.66 min (399.3 (M+1)). ¹H NMR(Methanol-d4) δ 2.2 (singlets, 3H), 2.41 and 2.45 (singlets, 3H),3.6-3.85 (m, 4H), 4.99 and 5.05 (singlets, 1H), 7.09 (br s, 1H), 7.39(d, 2H, j=8 Hz), 7.77 and 7.87 (d, 2H, j=8 Hz).

6.10. Synthesis ofN′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)benzohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(150 mg, 0.65 mmol) was suspended in methanol (3 ml) and water (1 ml).Benzoic acid hydrazide (102 mg, 0.75 mmol, 1.2 eq.) and hydrochloricacid (one drop, 12 N) were added, and the suspension was stirred at 50°C. for 18 hours. LCMS analysis indicated the formation of the productand the absence of starting material. The homogeneous reaction mixturewas cooled to room temperature and concentrated in vacuo. C-18Reverse-Phase SPE (10 g Alltech Hi-load C18, gradient from water to 20%methanol/water) to yield the product, a mixture of approximately 1:1 E:Zisomers, as a colorless solid: 193 mg (85%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.49 min (349.2 (M+1)). ¹H NMR (Methanol-d4) δ 2.2 (singlets, 3H),2.42 and 2.45 (singlets, 3H), 3.6-3.85 (m, 4H), 5.11 and 5.14 (singlets,1H), 7.30 (br s, 1H), 7.40-7.7 (m, 4H), 7.80 and 7.95 (m, 2H), 8.1 (brs, 1H).

6.11. Synthesis of (E)-ethyl2-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)hydrazinecarboxylate

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(150 mg, 0.65 mmol) was suspended in methanol (3 ml) and water (1 ml).Ethyl carbazate (78 mg, 0.75 mmol, 1.2 eq.) and hydrochloric acid (onedrop, 12 N) were added, and the suspension was stirred at 50° C. for 18hours. LCMS analysis indicated the formation of the product and theabsence of starting material. The reaction mixture was cooled to roomtemperature, concentrated in vacuo, and diluted with acetone. Theresulting white precipitate was collected and washed with acetone toyield the product, one apparent isomer, as a white solid: 96 mg (47%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 2-20% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.25 min (317.35 (M+1)). ¹H NMR (Methanol-d4) δ 1.36 (t, 3H, j=8Hz), 2.28 (s, 3H), 2.42 and 2.45 (singlets, 3H), 3.60-3.85 (m, 4H), 4.34(dd, 2H, j=8 Hz), 5.08 (s, 1H), 7.27 (s, 1H).

6.12. Synthesis of(E)-N′-(1-(4-((1R,2S,3R)-1.2.3.4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)nicotinohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(215 mg, 0.93 mmol) was suspended in methanol (3 ml) and water (1 ml).Nicotinic acid hydrazide (137 mg, 1.0 mmol, 1.1 eq.) and hydrochloricacid (one drop, 12 N) were added, and the suspension was stirred at 50°C. for 48 hours. LCMS analysis indicated the formation of the productand the absence of starting material. The reaction mixture was cooled toroom temperature, and partially concentrated in vacuo. The resultingwhite precipitate was collected and washed with water to yield theproduct, one apparent isomer, as a white solid: 311 mg (95%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.22 min (350.27 (M+1)). ¹H NMR (DMSO-d₆) δ 2.37 (s, 3H),3.60-3.85 (m, 4H), 4.40 (m, 2H), 4.71 (m, 1H), 5.01 (m, 2H), 5.16 (m,1H), 7.25 (br, 1H), 7.64 (br, 1H), 8.35 (br, 1H), 8.80 (br, 1H), 9.14(br, 1H).

6.13. Synthesis of3-chloro-N′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)benzohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(194 mg, 0.84 mmol) was suspended in ethanol (4 ml) and water (1 ml).3-chlorobenzoic acid hydrazide (170 mg, 1.0 mmol, 1.2 eq.) andhydrochloric acid (one drop, 12 N) were added, and the suspension wasstirred at 50° C. for 48 hours. LCMS analysis indicated the formation ofthe product and the absence of starting material. The reaction mixturewas cooled to room temperature, and partially concentrated in vacuo. Theresulting white precipitate was collected and washed with ethanol toyield the product, as a ^(˜)3:1 E:Z mixture, as a white solid: 108 mg(33%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.63 min (383.23 (M+1)). ¹H NMR (Methanol-d4) δ 2.44 (s, 3H),3.60-3.90 (m, 4H), 5.12 (s, 1H), 7.29 (s, 1H), 7.65 (m, 2H), 8.04 (m,2H).

6.14. Synthesis of(E)-4-fluoro-N′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)benzohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(172 mg, 0.74 mmol) was suspended in ethanol (4 ml) and water (1 ml).4-fluorobenzoic acid hydrazide (131 mg, 0.85 mmol, 1.1 eq.) andhydrochloric acid (one drop, 12 N) were added, and the suspension wasstirred at 55° C. for 48 hours. LCMS analysis indicated the formation ofthe product and the absence of starting material. The reaction mixturewas cooled to room temperature, and partially concentrated in vacuo. Theresulting white precipitate was collected and washed with ethanol toyield the product, as one apparent isomer, as a white solid: 97 mg(35%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.55 min (367.24 (M+1)). ¹H NMR (Methanol-d4, one drop DCl) δ 2.55(s, 3H), 3.60-3.90 (m, 4H), 5.22 (s, 1H), 7.30 (m, 2H), 7.54 (s, 1H),8.08 (m, 2H).

6.15. Synthesis of(E)-6-amino-N′-(1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)nicotinohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(115 mg, 0.50 mmol) was suspended in ethanol (4 ml) and water (1 ml).Substituted hydrazide (91 mg, 0.6 mmol, 1.2 eq.) and hydrochloric acid(one drop, 12 N) were added, and the suspension was stirred at 55° C.for 48 hours. LCMS analysis indicated the formation of the product andthe absence of starting material. The reaction mixture was cooled toroom temperature, and partially concentrated in vacuo. The resultingwhite precipitate was collected and washed with ethanol to yield theproduct, as one apparent isomer, as a white solid: 136 mg (75%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM ammoniumacetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.15 min (365.32 (M+1)). ¹H NMR (Methanol-d4, one drop DCl) δ 2.58(s, 3H), 3.60-3.90 (m, 4H), 5.22 (s, 1H), 7.17 (m, 1H), 7.54 (m, 1H),8.44 (m, 1H), 8.68 (m, 1H).

6.16. Synthesis of(E)-N′-(1-(4-((1R,2S3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)isonicotinohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(168 mg, 0.73 mmol) was suspended in ethanol (4 ml) and water (1 ml).Isonicotinic hydrazide (110 mg, 0.80 mmol, 1.1 eq.) and hydrochloricacid (one drop, 12 N) were added, and the suspension was stirred at 55°C. for 24 hours. LCMS analysis indicated the formation of the productand the absence of starting material. The reaction mixture was cooled toroom temperature, and partially concentrated in vacuo. The resultingwhite precipitate was collected and washed with ethanol to yield theproduct, as one apparent isomer, as a white solid: 136 mg (75%).

LCMS: Sunfire C-18 column, 4.6×50 mm; 10-90% in water (10 mM AmmoniumAcetate) over 2.5 min; flow rate=3.5 ml/min; Detection 220 nm; Retentiontime: 0.15 min (365.32 (M+1)). ¹H NMR (Methanol-d4, one drop DCl) δ 2.63(s, 3H), 3.60-3.90 (m, 4H), 5.12 (s, 1H), 7.58 (s, 1H), 8.63 (d, 2H, j=8Hz), 9.14 (d, 2H, j=8 Hz).

6.17. Synthesis of(E)-N′-(1-(4-((1R,2S,3)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethylidene)biphenyl-3-carbohydrazide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(315 mg, 1.36 mmol) and biphenyl-3-carbohydrazide (360 mg, 1.81 mmol)were suspended in DMSO (2 ml). Concentrated hydrochloric acid (twodrops) was added, and the suspension was stirred at 40° C. for 5 hours.LCMS analysis indicated the formation of the product and the absence ofstarting material. The reaction mixture was cooled to room temperature,diluted with methanol and purified by reverse phase HPLC (10 mMNH₄OAc/acetonitrile). Two fractions (E and Z isomers) of the desiredmass were collected separately and lyophized. Fraction one afforded awhite solid, 95 mg (16%). Fraction two was a white solid, 82 mg (14%).

Fraction one: Analytical HPLC Zorbax C-8 column, 4.6×150 mm; SolventA=10 mM ammonium acetate; Solvent B=MeCN; 5% B at 0 min, 5% B at 1 min,90% B at 3 min, 4 min stop; flow rate=3 ml/min; Detection 220 nm;Retention time: 2.9 min (note: contains ^(˜)5% of the other isomer).M+H=425.28. ¹H NMR (DMSO-d6 with 2 drops D₂O) δ 2.3 (singlet, 3H),3.3-3.7 (m, 4H), 4.9 (m, 1H), 7.19 (s, 1H), 7.37 (m, 1H) 7.47 (m, 2H),7.67 (m, 3H), 7.85-7.92 (m, 2H) and 8.15 (s, 1H). HSQC of the samesample correlated the proton signal at 2.3 (CH₃) with a carbon signal at20 ppm.

Fraction two: Analytical HPLC Zorbax C-8 column, 4.6×150 mm; SolventA=10 mM ammonium acetate; Solvent B=MeCN; 5% B at 0 min, 5% B at 1 min,90% B at 3 min, 4 min stop; flow rate=3 ml/min; Detection 220 nm;Retention time: 2.963 min (note: contains ^(˜)6% of the other isomer).M+H=425.28. ¹H NMR (DMSO-d₆ with 2 drops D₂O) δ 2.4 (singlet, 3H),3.4-3.6 (m, 4H), 4.77 and 4.86 (broad singlets, combined=1H), 6.9 and7.1 (broad singlets, combined=1H), 7.40 (m, 1H) 7.50 (m, 2H), 7.61 (m,1H), 7.73 (m, 2H), 7.87 (m, 2H) and 8.10 (s, 1H). HSQC of the samesample correlated the proton signal at 2.4 (CH₃) with a carbon signal at13 ppm.

6.18. Synthesis ofN-hydroxy-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazole-2-carboxamide

1-[4-((1R,2S,3R)-1,2,3,4-Tetrahydroxy-butyl)-1H-imidazol-2-yl]-ethanone(18 g, 78.3 mmol) was suspended in dichloroethane (160 ml) and2,2-dimethoxy propane (160 ml). 4-toluenesulfonic acid (3 g) was addedand the mixture stirred at 70° C. for 18 hours. The reaction was dilutedwith dichloromethane and washed with water, 5% bicarbonate, brine andthen dry loaded onto SiO₂. Purification by flash chromatography(hexane/ethyl acetate) afforded1-(4-((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolan)-5-yl)-1H-imidazol-2-yl)ethanoneas a colorless oil (18.8 g, 60.6 mmol, 77%; M+H calc: 311.4, obs:311.3).

The product obtained above (20 g, 64.5 mmol) was dissolved in DMF. K₂CO₃was added (12.5 g, 90.3 mmol) followed by benzyl bromide (10.7 ml, 90.3mmol). The reaction was heated at 50° C. for 18 h. LC/MS analysisindicated starting material remained. An additional portion of benzylbromide (5 ml, 42 mmol) was added and the temperature increased to 60°C. After 3 hours the reaction was quenched with cold water and extractedwith ethyl acetate. The organic extracts were washed with water, thenbrine, dried over sodium sulfate, and loaded onto silica gel. Flashchromatography (20 to 40% ethyl acetate in hexane) afforded1-(1-benzyl-4-((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolan)-5-yl)-1H-imidazol-2-yl)ethanone(16.1 g, 62%).

The intermediate obtained (13 g, 32.5 mmol) was dissolved in dioxane(120 ml) and treated with NaOH (13.2 g) dissolved in commercial bleach(200 ml, 6% NaOCl). After 2 h of vigorous stirring, the reaction wasextracted with ethyl acetate. Organic extracts were washed with brinethen dried over celite. Filtration and evaporation afforded a solid thatwas further dried in vacuo to afford1-benzyl-4-((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolan)-5-yl)-1H-imidazole-2-carboxylicacid (13 g, quantitative yield, M+H calc: 403.2, obs: 403.2).

The product obtained above (600 mg, 1.49 mmol), O-tritylhydroxylamine(820 mg, 2.98 mmol), EDAC (430 mg, 2.24 mmol) and HOBt (305 mg, 2.24mmol) were combined with DMF (8 ml) and triethylamine (622 μl, 4.47mmol). The reaction was stirred at ambient temperature for 22 h,concentrated and then loaded onto silica using DCM/MeOH. Flashchromatography (MeOH/DCM) afforded1-benzyl-4-((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolan)-5-yl)-N-(trityloxy)-1H-imidazole-2-carboxamide(480 mg, 0.73 mmol, 49%, M+H calc: 660.3, obs: 660.4).

The product obtained above (480 mg, 0.73 mmol) was dissolved in ethanol(50 ml). Pd(OH)₂ (500 mg, 20% on carbon, wet) was added and the reactionstirred under H₂ (65 psi) for 18 h and filtered. Ethanol was removed invacuo. The residue was dissolved in DCM and purified by flashchromatography (MeOH/DCM) to affordN-hydroxy-4-((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolan)-5-yl)-1H-imidazole-2-carboxamide(150 mg, 0.46 mmol, 63%, M+H calc: 328.1, obs: 328.3).

The product obtained above (150 mg, 0.46 mmol) was dissolved in acetone(8 ml) and water (8 ml). The reaction was cooled to an internaltemperature −15° C. using a dry ice/acetone bath. Concentrated HCl (3ml) was added at a rate such that the internal temperature remainedbelow −10° C. The cold bath was removed and the reaction stirred atambient temperature for 3 hours, at 4° C. for 18 h and again at ambienttemperature for 7 hours. After removal of the acetone and some water invacuo, a precipitate formed. Dioxane (20 ml) was added followed by THF(10 ml). The solid was isolated by filtration, washed with THF/dioxaneand dried in vacuo to affordN-hydroxy-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazole-2-carboxamideas the hydrochloride salt (98 mg, 0.40 mmol, 87%).

Mass spec.: M+H calc: 248.1, obs: 248.2. Analytical HPLC: LunaPheny-Hexyl, 5 um, 4.6×50 mm, isocratic 10 mM ammonium acetate with 1%acetonitrile, flow rate=3 ml/min, 220 nm detection, retention time=0.245min. ¹H NMR (DMSO-d6) δ 3.37-3.64 (m, 4H), 4.96 (broad singlet, 1H),7.47 (s, 1H), 11.9 (broad singlet, 1H).

6.19. Measuring Effects on Lymphocytes in Mice

Compounds were administered by oral gavage or in drinking water. Fororal dosing experiments, compounds were resuspended from crystals at 10mg/ml in vehicle (e.g., water). Mice (F1 hybrids of 129/B6 strain) weregavaged with a single 100 mg/kg dose of compound (equivalent to 100 mpkof the free base for each compound) or a vehicle-only control, andreturned to their cages. Mice were anesthetized using isofluoraneeighteen hours after dosing and tissues were collected for analysis asdescribed below. For drinking water studies, compounds were dissolved at50 mg/L in acidified water (pH=2.8) containing 10 g/L glucose. The micewere allowed free access to compound-containing water (or glucosesolution as a control) for 72 hours. At the end of 72 hours, tissueswere collected for analysis.

CBC measurements were obtained as follows. Mice were anesthetized withisofluorane and blood was collected from the retroorbital plexus intoEDTA blood collection tubes (Capiject-MQK, Terumo Medical Corp., Elkton,Md.). Automated CBC analysis was performed using a Cell-Dyn 3500 (AbbottDiagnostics, Abbott Park, Ill.) or a HemaVet 850 (Drew Scientific, Inc.,Oxford, Conn.) instrument.

Flow cytometry (FACS) measurements were obtained as follows. Twenty fiveμl whole blood was lysed by hyoptonic shock, washed once in 2 ml FACSwash buffer (FWB: PBS/0.1% BSA/0.1% NaN₃/2 mM EDTA) and stained for 30minutes at 4° C. in the dark with a combination offluorochrome-conjugated antibodies diluted in 50 μl FWB. After staining,the cells were washed once with 2 ml FWB and resuspended in 300 μl FWBfor acquisition.

Standard procedures for non-sterile removal of spleen and thymus werefollowed. Organs were dispersed into single-cell suspensions by forcingthe tissue through a 70 μm cell strainer (Falcon, Becton DickinsonLabware, Bedford, Mass.). For FACS analysis, RBCs were lysed byhypotonic lysis, washed, and 1×10⁶ cells were incubated with 10 μlanti-CD16/CD32 (Fc Block™, BD-PharMingen, San Diego, Calif.) (1/10dilution in FWB) for 15 minutes at 4° C. The cells were stained with acombination of fluorochrome-conjugated antibodies diluted in 50-100 μlFWB, added directly to the cells in Fc Block, for 30 minutes at 4° C. inthe dark. After staining the cells were washed once with 1 ml FWB, andresuspended in 300 μl FWB for acquisition. All antibodies were purchasedfrom BD-PharMingen, San Diego, Calif. unless otherwise specified.Samples were analyzed using a FACSCalibur flow cytometer and CellQuestPro software (Becton Dickinson Immunocytometry Systems, San Jose,Calif.).

Antibody mixes used for the thymus were: TCRb APC Cy7; CD4 APC; CD8PerCP; CD69 FITC; and CD62L PEI. Antibody mixes used for spleen andblood were: B220 PerCP; TCRb APC; CD4 APC Cy7; CD8 PE Cy7; CD69 FITC;and CD62L PE.

6.20. Measuring Effects on S1P Levels in Mice

Levels of S1P in mouse (F1 hybrids of 129/B6 strain) spleen weremeasured using an adaptation of the radio-receptor binding assaydescribed in Murata, N., et al., Anal. Biochem. 282:115-120 (2000). Thismethod utilized HEK293F cells overexpressing Edg-1, one of the S1Preceptor subtypes, and was based on the competition of labeled S1P withunlabeled S1P in a given sample.

HEK293F cells were transfected with a pEFneo S1P receptor(Edg-1)-expression vector and a G418-resistant cell clone was selected.The Edg-1-expressing HEK293F cells were cultured on 12 multiplates inDMEM containing 5% (v/v) FBS in a humidified air:CO₂ (19:1) atmosphere.Twenty four hours before the experiment, the medium was changed to freshDMEM (without serum) containing 0.1% (w/v) BSA.

Eighteen hours after the test compound was administered, mice weresacrificed and their spleens were removed and frozen. S1P was obtainedfrom the frozen tissue using known methods. See, e.g., Yatomi, Y., etal., FEBS Lett. 404:173-174 (1997). In particular, 10 mouse spleens in 1ml ice cold 50 mM phosphate buffer (pH 7.5) containing 1 mM EGTA, 1 mMDTT and Roche complete protease inhibitors were homogenized three timesat one minute intervals on ice. The result is centrifuged at 2500 rpmand 4° C. for 10 minutes to remove cell debris. The supernatant was thenultracentrifuged at 45000 rpm and 4° C. in a 70Ti rotor for 1 hour topull down the membrane-associated proteins. The supernatant wasdiscarded, and the pellet was resuspended in minimal volume (^(˜)1 ml)of ice cold 50 mM phosphate buffer (pH 7.5) containing 1 mM EGTA, 1 mMDTT and 33% glycerol with Roche complete protease inhibitors present.The total protein concentration was measured using the Bradford assay.

S1P was extracted into chloroform/KCl/NH₄OH (pH^(˜)12), and the upperaqueous phase is kept. It was then extracted in chloroform/methanol/HCl(pH<1), and the lower organic phase was kept and evaporated to provideS1P, which was stored in a freezer until used. Just before the assay,the dried sample was dissolved by sonication in a binder bufferconsisting of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, and 0.4%(w/v) BSA.

The S1P content of a sample was measured by a radioreceptor-bindingassay based on a competitive binding of [³³P]S1P with S1P in the sampleon Edg-1-expressing cells. Edg-1-expressing HEK293F cells in confluent12 multiplates were washed twice with the ice-cold binding buffer andthen incubated with the same buffer containing 1 nM [³³P]S1P (about18,00 dpm per well) and increasing doses of authentic S1P or test samplein a final volume of 0.4 ml. The plates were kept on ice for 30 minutes,and the cells were washed twice with the same ice-cold binding buffer toremove unbound ligand. The cells were solubilized with a solutioncomposed of 0.1% SDS, 0.4% NaOH, and 2% Na₂CO₃, and the radioactivitywas counted by a liquid scintillation counter. The S1P content in theassay well was estimated by extrapolation from the standard displacementcurve. The content of S1P in the initial test sample(s) was calculatedby multiplying the value obtained from the standard curve by therecovery efficiency of S1P extraction and the dilution factor.

6.21. Compounds' Effects on Lymphocytes in Mice

Using the methods described above, the in vivo effects of variouscompounds were determined. As shown in FIGS. 1-3, when administered tomice (F1 hybrids of 129/B6 strain) in drinking water, both THI and arepresentative compound of the invention (Compound 1) diminishedlymphocyte egress from the thymus.

FIG. 4 shows the effects of vehicle control (drinking water), THI,compound 1, and Compound 2 on whole blood counts. Interestingly, the invivo effects reported by Pyne (WO 97/46543) for Compound 2 were notobserved.

6.22. Collagen-Induced Arthritis Model

Collagen-induced arthritis (CIA) is a widely used model of rheumatoidarthritis (RA), a disease of the joint caused by autoimmune andinflammatory processes. See generally, Wooley P. H. et al., J. Immunol.135(4):2443-2451; A. Persidis, Nature Biotechnology 17:726-728; CurrentProtocols in Immunology (John Wiley & Son, Inc. 1996). Early studiesestablished a hierarchy of responsiveness to CIA linked to certain H-2haplotypes. More recently, Campbell and coworkers re-evaluated CIA inC57BL/129sv (H-2b) mice and found that mice derived from C57B6background can develop CIA. Campbell I. K, et al. Eur. J. Immunol. 30:1568-1575 (2000).

Here, collagen for injection, 2 mg/ml chicken collagen type II (CII)(Sigma) was dissolved in 10 mM acetic acid by stirring overnight at 4°C. Complete Freund's adjuvant (CFA) was purchased ready-made (Sigma).Using a glass syringe, CII was emulsified in an equal volume of CFA justprior to immunization. To immunize mice, a glass syringe and 26 G needlewere used, and mice were injected with CII/CFA emulsion intradermally atthe base of the tail. CFA and 100 μg of chicken CII in a total volume of50 μl CFA were used for each injection. A booster immunization of 100 μgof CII emulsified in CFA was given via the same route 3 weeks after theprimary immunization.

Injection of the collagen led to footpad and joint swelling of mice.Mice were inspected every two to three days for the onset of arthritis,which was assessed by combination of caliper measurements of thethickness of hind footpad and visual evaluation of the affected jointsof hind legs. The progression of CIA was followed for 10 weeks after theinitial onset of the disease, after which time the disease was assessedby histology of the joints. Mice were considered negative for arthritisif they did not develop CIA within 150 days of type II collagenimmunization.

The presence or absence of arthritis in mice was determined by anestablished visual scoring system. In mouse CIA, any or four paws can beaffected. The inflammation at its peak extends from ankle all the waythrough the digit, and is characterized by extreme swelling anderythema. Once arthritis appeared, each paw was examined 2 to 3 times aweek. To evaluate the severity of the inflammation, the widely usedvisual scoring of 0 to 4 was used, wherein: 0=normal, no evidence oferythema and swelling; 1=erythema and mild swelling confined to themid-foot or ankle joint or individual digits; 2=erythema and mildswelling extending to the ankle and the mid-foot or swelling in morethan one digit; 3=erythema and moderate swelling extending from theankle to the metatarsal joints; and 4=erythema and severe swellingencompassing the ankle, foot and digits.

6.23. Effect in Collagen-Induced Arthritis Model

The effect of a compound of the invention in the CIA model wasdetermined using 30 mice (F1 hybrids of 129/B6 strain) in the modeldescribed above. The mice were randomly divided into two blinded groups,which received 0 mpk (vehicle control) or 100 mpk of the compound. Thevehicle was sterile, distilled water. The vehicle control was dosed at10 μl/g body weight.

Dosing was carried out once a day by oral gavage. It began three daysprior to initiation of the CIA experiment, and continued throughout theduration of the experiment. FIG. 5 shows the effect of the compound onCIA over time, wherein the cumulative score is the sum of the fore-limband ankle scores.

6.24. The Effect of a Compound in Monkeys

The effect of a compound of the invention in twenty non-naïve, male,cynomolgus monkeys was investigated by Covance Research Products Inc.(Alice, Tex.). Each animal was identified with an individually numberedtattoo. The animals were acclimated for approximately 11 days prior todose administration. At the time of dose administration, animals wereconsidered to be young adult/adult in age.

During acclimation and the test period, animals were housed inindividual cages. Animals were not commingled for at least 48 hoursafter dose administration to allow monitoring of any vehicle- or testarticle-related effects. Animals had access to non-certified primatediet ad libitum, except as specified for dose administration. Fruits andother treats were also provided, as appropriate, during non-fastedperiods. Water was provided ad libitum.

The tested compound was stored protected from light in a sealedcontainer on desiccant under ambient conditions (approximate roomtemperature). Distilled water was supplied by Covance for oraladministration to animals in the vehicle control group. The test articledose formulations for the test were prepared on the day ofadministration. For each group, an appropriate quantity of the testarticle was weighed and an appropriate volume of either distilled water.

All animals were fasted overnight prior to dosing through approximately4 hours postdose. Individual doses were calculated based on body weightstaken on the day of dose administration.

The oral dose was administered via nasogastric intubation. Prior towithdrawing the gavage tube, the tube was flushed with approximately 5ml of water. For hematology analysis, blood (approximately 0.5 ml) wascollected from each animal predose (Day −7), predose (Day −3), and at 8,16, 24, 32, and 48 hours postdose. The whole blood hematology tests wereperformed using the fresh samples obtained on the day of collection.

As shown in FIG. 6, a single oral dose of the compound had a significanteffect on the monkeys' white blood cell and lymphocyte counts, whenmeasured 32 hours after dosing.

All cited publications, patents, and patent applications are hereinincorporated by reference in their entireties.

1. A method of treating or managing rheumatoid arthritis, whichcomprises administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is loweralkyl; R₃ is hydrogen, OR_(3A), NHC(O)R_(3A) or NHSO₂R_(3A); R₉ ishydrogen or CH₂OH; and each R_(3A) is independently hydrogen or loweralkyl.
 2. The method of claim 1, wherein the compound is of the formula:


3. The method of claim 2, wherein R₃ is OR_(3A).
 4. The method of claim3, wherein R_(3A) is hydrogen.
 5. A method of treating or managingrheumatoid arthritis, which comprises administering to a patient in needthereof a therapeutically effective amount of(E)-1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)-ethanoneoxime.